MOLECULAR AND CELLULAR BIOLOGY, Dec. 1995, p. 7106–7116 Vol. 15, No.

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Copyright q 1995, American Society for Microbiology

Degradation of the Proto-Oncogene Product c-Fos by the Ubiquitin

Proteolytic System In Vivo and In Vitro: Identification and
Characterization of the Conjugating Enzymes
ILANA STANCOVSKI,1 HEDVA GONEN,1 AMIR ORIAN,1 ALAN L. SCHWARTZ,2
1
AND AARON CIECHANOVER *

Department of Biochemistry and Rappaport Institute for Research in the Medical Sciences, Faculty of Medicine,
Technion-Israel Institute of Technology, Haifa 31096, Israel,1 and Division of Hematology-Oncology,
Children’s Hospital, and Washington University School of Medicine, St. Louis, Missouri 631102
Received 26 July 1995/Returned for modification 12 September 1995/Accepted 15 September 1995

The transcription factor c-Fos is a short-lived cellular protein. The levels of the protein fluctuate signifi-
cantly and abruptly during changing pathophysiological conditions. Thus, it is clear that degradation of the
protein plays an important role in its tightly regulated activity. We examined the involvement of the ubiquitin
pathway in c-Fos breakdown. Using a mutant cell line, ts20, that harbors a thermolabile ubiquitin-activating
enzyme, E1, we demonstrate that impaired function of the ubiquitin system stabilizes c-Fos in vivo. In vitro, we
reconstituted a cell-free system and demonstrated that the protein is multiply ubiquitinated. The adducts serve
as essential intermediates for degradation by the 26S proteasome. We show that both conjugation and
degradation are significantly stimulated by c-Jun, with which c-Fos forms the active heterodimeric transcrip-
tional activator AP-1. Analysis of the enzymatic cascade involved in the conjugation process reveals that the
ubiquitin-carrier protein E2-F1 and its human homolog UbcH5, which target the tumor suppressor p53 for
degradation, are also involved in c-Fos recognition. The E2 enzyme acts along with a novel species of
ubiquitin-protein ligase, E3. This enzyme is distinct from other known E3s, including E3a/UBR1, E3b, and
E6-AP. We have purified the novel enzyme ;350-fold and demonstrated that it is a homodimer with an
apparent molecular mass of ;280 kDa. It contains a sulfhydryl group that is essential for its activity,
presumably for anchoring activated ubiquitin as an intermediate thioester prior to its transfer to the substrate.
Taken together, our in vivo and in vitro studies strongly suggest that c-Fos is degraded in the cell by the
ubiquitin-proteasome proteolytic pathway in a process that requires a novel recognition enzyme.

The protein products of c-fos and c-jun function coopera-
tively as components of the mammalian transcription factor
AP-1 (reviewed in reference 1). The proteins form a stable
heterodimeric complex mediated via a leucine zipper associa-
tion that binds to a palindromic DNA sequence known as the
12-O-tetradecanoylphorbol 13-acetate (TPA)-responsive ele-
ment or the AP-1 binding site. Transcription of both c-fos and
c-jun is induced by TPA and other activators of protein kinase
C (1), resulting in increased biosynthesis of the two proteins.
Regulation of transcription and posttranslational modifica-
tions, such as phosphorylation, control the activity of AP-1 in
response to extracellular stimuli. The composition of the AP-1
complex changes from predominantly Jun homodimers prior
to induction to mostly Jun-Fos heterodimers following induc-
tion. Fos proteins (c-Fos, FosB, Fra-1, and Fra-2) are thought
to modulate AP-1 activity, as they cannot form homodimers
but only heterodimers with members of the Jun family proteins
(c-Jun, JunB, and JunD). The heterodimer has higher DNA-
binding and transcriptional activity than the Jun homodimer
(11). Inhibition of the activity of members of the Fos family,
either by expression of antisense transcript or following micro-
injection of specific antibodies, blocked the induction of cell
proliferation and cell cycle progression (29, 47). However,
gene disruption experiments (34, 63) suggest that the absence
of the c-fos gene is probably compensated for by other mem-
* Corresponding author. Mailing address: Department of Biochem-
istry, Faculty of Medicine, Technion-Israel Institute of Technology,
P.O. Box 9649, Haifa 31096, Israel. Phone: 972-4-295365, 972-4-
295379, or 972-4-295356. Fax: 972-4-513922 or 972-4-552296.
bers of the family. The viral counterparts of c-fos and c-jun
(v-fos and v-jun, respectively) have been identified as onco-
genes in transforming retroviruses (10, 37). In addition, dereg-
ulated expression of both c-Fos and c-Jun leads to neoplastic
transformation of rat fibroblasts (12, 54). It appears therefore
that the stability of Fos determines to a large extent the activity
of AP-1 and may be important for the proper control of cell
proliferation.
A series of previous observations suggest that at least two
proteolytic pathways may be involved in c-Fos breakdown. (i)
Using a cell-free system, Hirai and colleagues (27) and Carillo
and colleagues (5) demonstrated the involvement of calpains
in the breakdown of c-Fos and c-Jun. (ii) In a reticulocyte
lysate, c-Fos is slightly degraded in an ATP-dependent manner
(5, 8, 45). After interaction with phosphorylated c-Jun, c-Fos is
rapidly degraded by a trans degradation signal (45), but the
identity of the proteolytic system involved has remained ob-
scure. In the case of Jun-independent degradation, it has been
suggested that the process is mediated by the ubiquitin-pro-
teasome system: immunodepletion of the ubiquitin-activating
enzyme, E1, inhibited degradation of c-Fos, whereas addition
of purified enzyme restored activity (8). However, even in this
case, multiply ubiquitinated species of c-Fos, the essential in-
termediates in the process, have not been demonstrated, and
the enzymes involved in conjugation and degradation have not
been identified. Treier and colleagues suggested a role for the
ubiquitin pathway in regulating the stability of c-Jun (59) and
demonstrated that targeting is mediated via the d domain in
the c-Jun molecule. Interestingly, v-Jun, which is a stable pro-
tein, lacks the d domain and escapes ubiquitination and sub-

7106
VOL. 15, 1995
sequent degradation. The researchers proposed that this sta-
bilization plays a role in the oncogenicity of v-Jun. However,
Jariel-Encontre and colleagues have recently shown that ubi-
quitination is not required for degradation of the c-Jun pro-
tein, which can be degraded by the 26S proteasome without
prior ubiquitination (31).
The ubiquitin-dependent pathway plays an important role in
the degradation of short-lived regulatory proteins, such as cy-
clins (reviewed in reference 40), the tumor suppressor protein
p53 (9, 49, 50–53), and the MATa2 repressor (28). In addition,
the system is also involved in limited proteolysis, such as in the
maturation of p105, the precursor of the NF-kB transcriptional
activator (43), and probably in processing of antigens restricted
to class I major histocompatibility complex (MHC) molecules
(38). Degradation of a protein via the ubiquitin system involves
several discrete steps. In this process, ubiquitin is first activated
by the ubiquitin-activating enzyme E1. The activated molecule
is transferred to one of several ubiquitin carrier proteins (E2s,
or ubiquitin-conjugating enzymes [UBCs]), which transfers it
to a third enzyme, ubiquitin-protein ligase, E3. This enzyme
catalyzes the last step in the process, covalent conjugation of
ubiquitin to the protein substrate that bound to the ligase (7,
52). Following formation of a multiubiquitin chain, the tagged
protein is degraded by the 26S proteasome complex, with the
release of free and reutilizable ubiquitin (reviewed in refer-
ences 7, 14, and 32).
Using a cell line harboring a thermolabile E1 enzyme, we
demonstrate that c-Fos is stabilized at the restrictive temper-
ature. Dissection of the proteolytic pathway in a cell-free sys-
tem reveals that c-Fos, which is a short-lived protein in the cell,
is multiply ubiquitinated and that the high-molecular weight
conjugates serve as intermediates for proteolysis by the 26S
proteasome complex. We also demonstrate that the process is
mediated by a recently described ubiquitin carrier protein,
E2-F1 (2), and a novel, as yet unidentified, species of E3. The
E2-F1 enzyme can be functionally replaced by a recombinant
human homolog, UbcH5 (50). Both conjugation and degrada-
tion are strongly stimulated by c-Jun. Taken together, the data
strongly suggest that the ubiquitin pathway is involved in reg-
ulating the stability of the transcription factor c-Fos, both in
vivo and in vitro, and thus plays an important role in regulating
the cellular level of AP-1 complex and its transcriptional ac-
tivity.
MATERIALS AND METHODS
Cell lines. The Chinese hamster cell line ts20, a temperature-sensitive cell
cycle-arrested mutant, and its parental cell line E36 were previously described
(36). ts20 cells harbor a thermolabile ubiquitin-activating enzyme, E1, and ubi-
quitin conjugation is significantly decreased at the nonpermissive temperature.
Consequently, degradation of short-lived proteins in these cells is also impaired
(19). The ts20.E1.C2 (ts-E1) cell line (a generous gift from Patricia M. Handley-
Gearhart, Washington University School of Medicine [WUMS], St. Louis, Mo.)
was obtained by transfection of ts20 cells with a human wild-type E1 cDNA (21).
The B-cell lymphoma line C3.F6 (62) was obtained from Osami Kanagawa,
WUMS. Cells were grown as a monolayer at 308C (E36, ts20, and ts-E1) or in
suspension at 378C (C3.F6) in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% fetal calf serum (FCS). Transfections were carried out
by electroporation (64). The permissive and nonpermissive temperatures used
for the experiments with the various E1 cell lines were 30 and 39.58C, respec-
tively.
Plasmids. The cDNAs for rat c-fos in pSP65 and human papillomavirus type
16 E6 in pGEM-2 vectors have been described elsewhere (8, 9). The v-fos-
containing plasmid was constructed by replacement of the NcoI-AvrII fragment
from the c-fos cDNA in the pSP65 vector with the respective fragment from the
FBJ-2 vector (13). In vitro transcription-translation reactions for generation of
[35S]methionine-labeled proteins were performed in a TNT-coupled wheat germ
extract system (Promega) according to the manufacturer’s instructions. The
pKH6 vector (44, 45) containing a hexahistidine (6-His)-tagged human c-jun was
a generous gift from Dirk Bohmann (EMBL, Heidelberg, Germany). The bac-
c-Fos DEGRADATION BY THE UBIQUITIN SYSTEM 7107
terial expression plasmid encoding the UbcH5 (50) was obtained from Martin
Scheffner (Deutsches Krebsforschungszentrum, Heidelberg, Germany).
Degradation of c-Fos protein in vivo. Exponentially growing cells were seeded
at 5 3 105 cells per 65-mm dish. After 16 h, the medium was changed to DMEM
containing 0.5% FCS, and incubation was continued for additional 48 h. Induc-
tion of c-Fos was accomplished by the addition of DMEM containing 20% FCS
for 1 h. For labeling, cells were washed with methionine-free medium and
incubated at 308C for 15 min in the same medium containing 20% dialyzed FCS
and 300 mCi of [35S]methionine per ml. Labeling of the cells was continued for
additional 20 min at either 30 or 43.58C (pulse). Cultures were harvested (0 min)
or incubation was continued (following removal of the label; chase) at 30 or
39.58C for 30 and 60 min in a complete medium containing 4 mM unlabeled
methionine. Cells were washed twice in ice-cold phosphate-buffered saline (PBS)
and lysed in 1 ml of ice-cold RIPA buffer (PBS containing 1% Nonidet P-40,
0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 2 mM phenyl-
methylsulfonyl fluoride [PMSF], and 5 mg of aprotinin per ml). Following cen-
trifugation at 13,000 3 g for 30 min, aliquots from the supernatant that contained
equal amounts of protein were immunoprecipitated with anti-Fos antibody
SC-52 (Santa Cruz Biotechnology), resolved by SDS-polyacrylamide gel electro-
phoresis (PAGE), and analyzed by fluorography.
Fractionation of crude reticulocyte lysate, preparation of E1 and E2-F1 en-
zymes, and initial characterization of the Fos conjugating ligase E3. Reticulo-
cyte-rich blood was induced in rabbits by successive injections of phenylhy-
drazine, and reticulocyte lysate was prepared as described before (25). Lysates
were resolved by anion-exchange chromatography over DEAE-cellulose (What-
man) into unadsorbed material (fraction I) and high-salt eluate (fraction II) as
described before (25). E1 was purified to homogeneity from fraction II following
affinity chromatography over immobilized ubiquitin (25). E2-F1 was purified to
homogeneity from fraction I as described before (2). Recombinant UbcH5, the
human homolog of E2-F1 (50) (cDNA cloned into pET3a vector) was purified
from bacterial extracts (following induction) as follows. Extract (from 1 liter of
cultured cells) was chromatographed on DEAE anion-exchange resin. Fraction
I (;75 mg of protein) was concentrated (to ;2.5 ml) with a Centriprep 10
(Amicon) and resolved on a HiLoad Superdex 75 HR (16 by 600 mm) with a fast
protein liquid chromatography (FPLC) system (Pharmacia) in a buffer contain-
ing 20 mM Tris-HCl (pH 7.2), 2 mM dithiothreitol (DTT), and 150 mM NaCl
(buffer A). Fractions (2.4 ml) were collected, and the protein was visualized by
Coomassie brilliant blue staining after SDS-PAGE of aliquots from the various
fractions. Salt was removed by repeated concentration-dilution cycles with Cen-
tricon-10 microconcentrators (Amicon) and buffer A without NaCl. The UbcH5-
containing fractions were concentrated to ;100 ml and pooled. We obtained ;1
mg of .85% homogeneous protein. For initial characterization of the E3 en-
zyme, rabbit reticulocyte fraction II was precipitated by (NH4)2SO4 into fractions
IIA (0 to 38%) and IIB (42 to 80%). Fraction IIA (2 ml, 20 mg/ml) was loaded
onto a Superdex 200 HR gel filtration chromatography column (16 by 600 mm)
and resolved and treated as described for UbcH5. Conjugation of c-Fos was
determined in the presence of 2.5-ml aliquots as described below.
Purification of the E3 protein. (i) Anion-exchange chromatography. Fraction
IIA (200 mg) was loaded onto a Resource Q (Pharmacia) anion-exchange col-
umn (16 by 120 mm). The column was equilibrated with 20 mM Tris-HCl (pH
7.2), 180 mM KCl, and 1 mM DTT. After loading, the column was washed
thoroughly with the equilibrating buffer. Adsorbed proteins were eluted with a
500-ml linear salt gradient to 500 mM KCl. Fractions (20 ml) were collected, and
salt was removed with Centriprep-30 concentrators as described above. Fos-
conjugating activity (E3-Fos) was eluted at ;240 to 280 mM KCl.
(ii) Gel filtration chromatography. Pooled and concentrated fractions from
the first purification step were loaded onto a Superdex 200 HR gel filtration
column (16 by 600 mm), and proteins were resolved as described above. Frac-
tions containing E3-Fos activity (proteins eluted at ;280 kDa) were pooled and
dialyzed against a buffer containing 20 mM Tris-HCl (pH 7.2), 1 M (NH4)2SO4,
and 1 mM DTT (buffer B).
(iii) Hydrophobic chromatography. Pooled and dialyzed fractions from the
second purification step were loaded onto a Bio-Rad Econo-Pac Methyl-HIC
hydrophobic column (5-ml bed volume) equilibrated in buffer B. Unadsorbed
proteins were washed with buffer B. A 100-ml linear gradient [1.0 to 0.0 M
(NH4)2SO4] was used to elute bound proteins. Salt was removed and fractions
were concentrated as described above. E3-Fos activity was eluted at ;500 mM
salt.
(iv) Hydroxylapatite chromatography. A portion of the hydrophobic-chroma-
tography-eluted E3 was dialyzed against a buffer containing 10 mM KPi (pH 7.4)
and 1 mM DTT and loaded onto a Bio-Rad Econo-Pac HTP (hydroxylapatite;
5-ml bed volume) column equilibrated in the same buffer. Adsorbed proteins
were eluted with a 100-ml linear gradient to 600 mM KPi. E3-Fos activity was
eluted at ;320 to 410 mM KPi.
(v) Anion-exchange chromatography. The remaining portion of the hydropho-
bic-chromatography-eluted E3 was dialyzed against a buffer containing 20 mM
Tris-HCl (pH 7.2), 200 mM KCl, and 1 mM DTT and loaded onto a Mono Q
(Pharmacia) anion-exchange chromatography column (1-ml bed volume) equil-
ibrated in the same buffer. E3-Fos protein was eluted at ;300 to 340 mM KCl
with a 200 to 600 mM linear gradient.
The protein concentration was determined throughout the purification by the
Bradford method (3) with bovine serum albumin as a standard. Protein compo-
7108 STANCOVSKI ET AL. MOL. CELL. BIOL.

sition was determined by SDS-PAGE and Coomassie brilliant blue or silver

staining. c-Fos conjugation was determined as described below.
Conjugation and degradation of [35S]methionine-labeled proteins. [35S]me-
thionine-labeled substrates were generated in vitro in a coupled transcription-
translation reaction in wheat germ extract (TNT; Promega). Conjugation and
degradation assays were performed essentially as described before (9). Briefly,
the reaction mixture contained, in a final volume of 25 ml, crude reticulocyte
lysate (10 ml; ;1 mg of protein) or fraction II (;100 mg of protein) and purified
E2-F1 (0.2 mg), 40 mM Tris-HCl (pH 7.6), 5 mM MgCl2, 2 mM DTT, 5 mg of
ubiquitin, and [35S]methionine-labeled substrate (;30,000 cpm). In the conju-
gation assays, 1 mg of ubiquitin aldehyde (26) and 5 mM adenosine 59-O-(3-
thiotriphosphate) (ATP-g-S) (Boehringer, Mannheim, Germany) were added.
Reaction mixtures without ATP contained 0.5 mg of hexokinase and 10 mM
2-deoxyglucose (9). For degradation, ATP (0.5 mM) and the ATP-regenerating
system (10 mM phosphocreatine and 5 mg of creatine kinase) were added.
Conjugation reactions were incubated for 30 min at 378C, and degradation
reactions were carried out at the same temperature for 2 h. Following incubation,
reaction mixtures were resolved via SDS-PAGE, and the gels were fluoro-
graphed, dried, and exposed to BioMax X-Ray film (Kodak) or to a phosphor-
imaging plate scanner (Fuji, Tokyo, Japan) for quantitative evaluation. The
radioactivity incorporated into adducts was expressed as a percentage of total
radioactivity.
Degradation of c-Fos conjugates by the 26S proteasome. For analysis of the
role of the 26S proteasome complex in the degradation of c-Fos-ubiquitin con-
jugates, adducts were synthesized as described above in a 100-ml reaction mix-
ture. Following incubation, enzymes were inactivated by the addition of NaOH
to a final concentration of 0.1 N and further incubation for 10 min. The pH was
neutralized by the addition of HCl, and the conjugates were dialyzed at 48C
against a buffer containing 20 mM Tris-HCl (pH 7.2) and 2 mM DTT. Degra- FIG. 1. Metabolic stability of c-Fos in E1-defective cell line. Endogenous
dation of c-Fos conjugates was assayed in the presence of purified 26S protea- c-Fos protein was induced by 20% FCS, and cells were pulse-labeled with
some complex as described before (18, 56). Briefly, reaction mixtures contained, 35
[ S]methionine for 35 min as described in Materials and Methods. Inactivation
in a final volume of 50 ml, conjugates (;10,000 cpm), 40 mM Tris-HCl (pH 7.6), of E1 was carried out by incubating the cells at 43.58C for 20 min. Cell lysates
5 mM MgCl2, and 2 mM DTT. The 26S proteasome complex (1.5 mg) and 1 mM were prepared without further incubation (0 min) or after incubation at 30 or
ATP were added as indicated. Following incubation at 378C for 2 h, conjugate 39.58C for 30 and 60 min (chase). Immunoprecipitates with anti-Fos antibody
degradation was determined by quantifying the remaining adducts with a phos- were analyzed by SDS-PAGE and fluorography.
phorimaging plate scanner. In parallel, release of radioactivity into the trichlo-
roacetic acid-soluble fraction was also monitored. Since the preparation of the
conjugates also contains free, unreacted labeled c-Fos, degradation of an equal
amount of radiolabeled c-Fos was monitored in parallel.
Formation of 125I-ubiquitin thioester with E1, E2-F1, and E3-Fos. Ubiqui- E36 at both temperatures (;40 min; quantitative analysis not
tin-E1 and ubiquitin-E2 thioester adducts were generated and detected with
125
shown). Inhibition of E1 resulted in three- to fourfold prolon-
I-ubiquitin as described before (2). To obtain the E3-Fos-ubiquitin thioester, gation of the half-life of the protein (analysis not shown). The
E3 (5 mg) was added to a reaction mixture that also contained E1, E2-F1,
125
I-ubiquitin, and ATP. partial stabilization is due, most probably, to the leakiness of
Purification of recombinant His-Jun. The 6-His-c-Jun protein was purified the mutation and to the inability to inhibit completely the E1
from transfected HeLa or E36 cell lines with a nickel chelate column (Ni21- enzyme. This problem has previously been discussed in detail
nitrilotriacetic acid-agarose; Quiagen) as previously described (44, 45). (19) (see also below). Incubation of the cells at the nonper-
Preparation of methylated ubiquitin. Reductively methylated ubiquitin
(MeUb) was prepared from bovine ubiquitin (Sigma) by a previously described missive temperature did not affect viability (.95%) (19). To
procedure (24). Fluorescamine determination of free amino groups revealed that ensure that c-Fos stabilization was not due to a change in other
more than 95% of the moieties were blocked. factors or parameters in the mutant cell line, we used an
Preparation of cell extracts. Cells were washed twice in ice-cold PBS and E1-rescued cell line, ts-E1. This cell line was obtained by stable
incubated on ice for 15 min in a hypotonic lysis buffer (20 mM sodium-HEPES
[N-2-hydroxyethylpiperazine-N9-2-ethanesulfonic acid, pH 7.5], 10 mM KCl, 1.5 correction of the defect in the E1 enzyme following transfec-
mM MgCl2, 2 mM DTT; 2 ml for 5 3 108 cells). Lysis was completed with a tion with an expression vector carrying wild-type human E1
Dounce homogenizer and controlled under the microscope. Nuclei were re- cDNA (21). As depicted in Fig. 1, c-Fos was rapidly degraded
moved and cytosol was prepared as described before (15). Extracts contained ;5 in the ts-E1 cell line at the nonpermissive temperature, cor-
to 10 mg of protein per ml.
roborating the involvement of E1 in destabilizing the protein.
We noted the appearance of high-molecular-mass c-Fos-
RESULTS related proteins in the wild-type, mutant, and wild-type E1-
transfected cells at the low temperature and in the wild-type
Functional ubiquitin-activating enzyme E1 is essential for and transfected cells at the high temperature. These bands are
c-Fos degradation in vivo. To evaluate the potential role of the most probably ubiquitin conjugates of c-Fos, as they disappear
ubiquitin system in the turnover of the c-Fos transcription in the mutant cells at the high temperature. We were able to
factor in vivo, we used ts20 cells, a cell cycle-arrested mutant detect a slight increase in these labeled proteins following the
derived from the E36 cell line that harbors a thermolabile addition of the proteasome inhibitor MG115 (not shown; for
ubiquitin-activating enzyme, E1 (36). At the nonpermissive the inhibitor, see below). However, because of their low abun-
temperature, conjugation of ubiquitin is significantly dimin- dance, we could not detect them directly with antiubiquitin
ished. Consequently, the degradation of the bulk of short-lived antibody and Western immunoblot analysis. It is possible that,
proteins is also inhibited (19). We monitored the degradation unlike adducts of other proteins, such as those of the cyclin-
of c-Fos at both the permissive temperature (308C) and after a dependent kinase inhibitor p27 (42), the conjugates of c-Fos
shift to the restrictive temperature (39.58C). As shown in Fig. are more sensitive to isopeptidases and thus do not accumulate
1, the c-Fos protein was stable in the mutant cell line, ts20, at to a detectable level even when the proteasome is inhibited.
the restrictive temperature but was rapidly degraded at the Thus, at this point we can speculate on their identity but not
permissive temperature. Quantitative measurement of c-Fos prove it unambiguously.
radioactivity indicated that the half-life of the protein in ts20 It should be noted that stabilization of any single short-lived
cells at 308C is similar to that measured in the parental cell line protein in the E1 mutant cells does not unequivocally demon-
VOL. 15, 1995
FIG. 2. E2-F1- and UbcH5-dependent conjugation of ubiquitin to c-Fos.
Conjugation of ubiquitin to [35S]methionine-labeled c-Fos was monitored by
SDS-PAGE and fluorography as described in Materials and Methods. (A) Ubiq-
uitin conjugation to c-Fos in a crude reticulocyte lysate. Incubation was carried
out in the absence (lane 1) or presence (lane 2) of ATP-g-S. (B) Ubiquitin
conjugation to c-Fos in fraction II. Lane 1, fraction II; lane 2, fraction II plus
ubiquitin; lane 3, fraction II, ubiquitin, and purified E2-F1. (C) Ubiquitin con-
jugation to c-Fos in lymphocyte cell extract. Cytosolic cell extract was prepared
as described in Materials and Methods and resolved on DEAE-cellulose. The
reaction mixtures in the different lanes are the same as those in panel B. (D)
c-Fos conjugation in the presence of recombinant UbcH5. c-Fos was incubated
in the presence of reticulocyte fraction II and ubiquitin only (lane 1) or plus
purified E2-F1 (lane 2) or 0.5 mg of recombinant UbcH5 (lane 3). Ori, origin of
the gel; conj, conjugates.
strate direct involvement of the ubiquitin pathway in its pro-
teolysis. One can always argue that stabilization of the target
protein is due to an upstream effect that results from E1 inac-
tivation. However, such an effect is not known and cannot be
analyzed at our current state of knowledge. The in vivo effect
must therefore be supported by additional experimental evi-
dence derived from the establishment of a faithful cell-free
system.
Ubiquitination of c-Fos in vitro. Degradation of a protein
via the ubiquitin system is a complex process that involves
specific enzymatic components and primary proteolysis recog-
nition signals on the target substrate. In addition, targeting can
also involve ancillary proteins and secondary, posttranslational
modifications that render the substrate susceptible to recogni-
tion. Thus, the initial observation that E1 is involved in c-Fos
degradation in vitro (8) and in vivo (see above) still leaves
numerous important unresolved problems. The ubiquitin con-
jugates which are the essential intermediates in the process
have not been demonstrated. The conjugating enzymes have
not been identified, and their mechanism of action has not
been dissected. The proteolysis recognition signals of the sub-
strate are still obscure, and the potential role of c-Jun in tar-
geting c-Fos for conjugation has not been studied. Thus, it was
important to establish an in vitro system that will reproduce
faithfully the ubiquitin-mediated proteolytic process. As can be
seen in Fig. 2, c-Fos can generate high-molecular-mass cova-
lent conjugates with ubiquitin. In the experiment presented in
Fig. 2A, the c-Fos substrate was incubated in a reticulocyte
lysate in the absence (lane 1) and presence (lane 2) of ATP-
g-S. To enhance the formation of conjugates, the reaction
contained ATP-g-S (instead of ATP) and ubiquitin aldehyde.
ATP-g-S promotes conjugation by E1 but does not support
c-Fos DEGRADATION BY THE UBIQUITIN SYSTEM 7109
degradation by the 26S proteasome complex (35). Ubiquitin
aldehyde inhibits certain isopeptidases (26). Under these con-
ditions, the steady-state level of ubiquitin adducts increases,
facilitating their detection. Next, we tested the formation of
c-Fos conjugates in a fractionated lysate. As shown in Fig. 2B
(lane 1), incubation of c-Fos in the presence of fraction II does
not lead to formation of conjugates. Fraction II (the high-salt
eluate obtained during anion-exchange chromatography of cell
extracts) contains most of the conjugating enzymes and the 26S
protease. However, ubiquitin and certain ubiquitin-conjugat-
ing enzymes, E2s, are contained in fraction I, the material that
does not adsorb to the resin. Addition of ubiquitin to fraction
II had only a slight effect on the formation of conjugates (Fig.
2B, lane 2). Addition of homogeneously purified E2-F1 led to
a significant increase in conjugate formation (Fig. 2B, lane 3).
E2-F1 is a ubiquitin-conjugating enzyme involved in targeting
non-‘‘N-end rule’’ substrates (2), including the tumor suppres-
sor protein p53 (9). To extend these results to a more relevant
physiological system, extracts from C3.F6, a B-cell lymphoma
cell line, were fractionated in a similar manner. As can be seen
in Fig. 2C, the formation of c-Fos conjugates is similar to that
observed in the reticulocyte lysate. E2-F1 is a rabbit enzyme,
homologous to several evolutionarily conserved members of
the E2 family. It is similar to Saccharomyces cerevisiae UBC4
and UBC5 (55), Drosophila melanogaster UbcD1 (58), Triticum
aestivum UBC8 (17), and several enzymes from Arabidopsis
thaliana (16). It has been shown that the wheat germ enzyme
accounts for most of the conjugating activity in extracts derived
from the plant (17). Recently, UbcH5, a human member of this
subfamily, was characterized and cloned (50). The enzyme is
also involved in targeting of p53 (50). Bacterially expressed
UbcH5 could functionally replace E2-F1 protein in promoting
c-Fos conjugation (Fig. 2D). Since UbcH5, UBC8, and E2-F1
can also catalyze E6-dependent conjugation of p53, it is pos-
sible that members of this highly conserved E2 family have a
similar function in the cell and play an important role in the
regulation of stability of many cellular proteins.
Influence of MeUb on c-Fos conjugation. To further evalu-
ate the role of ubiquitin in c-Fos conjugation, we used a de-
rivative of the protein in which the amino groups had been
blocked by reductive methylation, methylated ubiquitin
(MeUb). It was previously shown that MeUb is efficiently li-
gated to protein substrates but cannot form polyubiquitin
chains (24). Thus, MeUb can be used as a chain terminator in
the process of ubiquitin conjugation. The effect of different
concentrations of MeUb on c-Fos conjugation was examined in
the experiment shown in Fig. 3. [35S]methionine-labeled c-Fos
was subjected to conjugation in a partially purified in vitro
system containing E1, E2-F1, and fraction IIA as a source of
E3 in the presence of 5 mg of ubiquitin. As can be seen in Fig.
3, increasing concentrations of MeUb significantly inhibited
c-Fos conjugate formation. From quantitative evaluation of the
results presented in Fig. 3, the concentration of MeUb re-
quired for 50% inhibition was threefold higher than the cor-
responding concentration of ubiquitin, as was described previ-
ously (23).
Effect of c-Jun on ubiquitin-dependent degradation and con-
jugation of c-Fos. It has been shown previously that c-Fos
degradation is targeted by a phosphorylation-dependent signal
on c-Jun (45). The researchers proposed a model in which a
signal-induced dephosphorylation of c-Jun (for example, fol-
lowing treatment of cells with TPA) transiently stabilizes c-
Fos–c-Jun dimers in stimulated cells. After withdrawal of the
stimulating agent and phosphorylation of c-Jun, AP-1 activity
decreases as a result of protein degradation (45). However, the
system that mediates the process and the mechanism(s)
7110 STANCOVSKI ET AL.
FIG. 3. Inhibition of ubiquitin conjugation to c-Fos by MeUb. [35S]methi-
onine-labeled c-Fos was subjected to ubiquitin conjugation in a reticulocyte
lysate as described in Materials and Methods. MeUb was added at the indicated
concentrations, and the reaction mixtures were resolved via SDS-PAGE. The
chart represents quantitative analysis of the data by phosphorimaging plate
scanner analysis.
through which c-Jun targets c-Fos for degradation have not
been identified. We investigated the possibility that c-Jun is
involved in the conjugation of ubiquitin to c-Fos, leading to
subsequent degradation of the protein. trans recognition has
been previously described in the case of the tumor suppressor
p53: the protein is targeted for ubiquitin conjugation and deg-
radation by the human papillomavirus E6 oncoprotein (50).
We therefore purified 6-His-tagged c-Jun (His-Jun) (44, 45)
and assessed its effect on in vitro degradation of c-Fos. As
demonstrated in Fig. 4A, and in agreement with previous data
(45), increasing concentrations of purified His-Jun target la-
beled c-Fos for degradation. In order to identify the step in the
degradation pathway that is affected by c-Jun, we analyzed the
effect of purified His-Jun on the conjugation of ubiquitin to
c-Fos. The reaction was carried out in a partially purified
system containing labeled c-Fos, ubiquitin-activating enzyme
E1, ubiquitin carrier protein E2-F1, and fraction IIA as a
source of ubiquitin-protein ligase E3. It is clear that addition of
His-Jun increases the formation of high-molecular-weight c-
Fos conjugates (Fig. 4B). It should be noted that ubiquitin is
conjugated to the labeled substrate even in the absence of
His-Jun (Fig. 4B, lane 2). However, the efficiency of degrada-
tion of c-Fos is significantly lower in the absence of c-Jun. It is
possible that the detection of the c-Jun effect on conjugate
formation is more difficult than monitoring its effect on deg-
radation because the adducts are also metabolized by isopep-
tidases not inhibited by ubiquitin aldehyde. Alternatively, it is
also possible that Jun affects other, as yet unidentified, ubiqui-
tin-independent proteolytic pathway(s) (see above). Stabiliza-
tion of c-Fos in E1-inactivated cells makes the possibility that
such putative pathways are operating in the cell rather unlikely.
The 26S proteasome is required for c-Fos degradation. The
results described above demonstrate that the c-Fos protein
forms conjugates with ubiquitin, suggesting a role for ubiq-
uitin-mediated proteolysis of this short-lived transcription fac-
MOL. CELL. BIOL.
FIG. 4. Effect of c-Jun on degradation and conjugation of c-Fos. (A) c-Fos
degradation. Degradation of [35S]methionine-labeled c-Fos was assayed in a
reticulocyte lysate as described in Materials and Methods. Control reactions
without ATP were incubated at either 08C (lane 1) or 378C (lane 2). Lanes 3 to
6, reaction mixtures containing ATP were incubated at 378C in the absence (lane
3) or presence (lanes 4 to 6) of the indicated amounts of His-tagged c-Jun. (B)
c-Fos conjugation. Conjugation of labeled c-Fos was carried out essentially as
described in Materials and Methods in the presence of a partially purified system
containing E1, E2-F1, and fraction IIA in the absence (lane 1) or presence of
ATP (lanes 2 to 4) and the indicated amounts of His-Jun (lanes 3 and 4). Ori,
origin of the gel; conj, conjugates.
tor. However, it was not clear that the modification leads to the
degradation of the protein and that the protease involved is
indeed the 26S proteasome complex. Previous studies have
shown that a peptide-aldehyde, MG115 (carbobenzoxyl-leuci-
nyl-leucinyl-norvalinal-H), is a potent and rather specific in-
hibitor of the 20S proteasome (43, 48). In vitro-translated
[35S]methionine-labeled c-Fos was incubated in cytosolic cell
extracts at 378C for 2 h (Fig. 5). Potential calpain-dependent
proteolysis was inhibited by treatment of the extract with the
calcium chelator EGTA (ethylene glycol tetraacetic acid) at 3
mM (5) (MgCl2 was added to the reaction mixture at a con-
centration of 10 mM). Addition of MG115 markedly inhibited
c-Fos degradation (Fig. 5, lanes 4 and 5). It should be noted
that in the presence of ATP (Fig. 5, lanes 3 to 5), a higher-
molecular-mass form of c-Fos accumulated. This change is due
FIG. 5. Inhibition of the 26S proteasome blocks c-Fos degradation. Degra-
dation of [35S]methionine-labeled c-Fos was carried out in a lymphocyte extract
for 2 h as described in Materials and Methods. Lanes 1 and 2, incubations at 0
and 378C, respectively, in the absence of ATP. Lanes 3 to 5, incubations at 378C
in the presence of ATP. MG115 was added at the indicated concentrations.
VOL. 15, 1995 c-Fos DEGRADATION BY THE UBIQUITIN SYSTEM 7111

FIG. 6. Identification of the E3 enzyme involved in c-Fos conjugation. (A) c-Fos conjugation. Fraction IIA was resolved via gel filtration chromatography over a
Superdex 200 HR column as described in Materials and Methods. E3 activity was monitored in the presence of E1, E2-F1, ubiquitin, ATP-g-S, and ubiquitin aldehyde,
in the presence of aliquots derived from the resolved fractions. (B) p53 conjugation. Conjugation of p53 was monitored as described in the legend to panel A in the
presence of in vitro-translated, unlabeled E6. Lane A, [35S]methionine-labeled substrates incubated in the presence of purified E1 and E2-F1. Lane B, control reactions
incubated as described for lane A but with the addition of 10 mg of unresolved fraction IIA. Other lanes, reaction mixtures incubated in the presence of E1, E2-F1,
and aliquots from the resolved fractions indicated above the lanes. The positions of molecular size markers and labeled substrates are marked. Arrows represent
fractions with the highest activity. Ori, origin of the gel; conj, conjugates.

to a posttranslational modification, most probably phosphory-
lation, that can significantly alter the migration of c-Fos in
SDS-PAGE (8, 12). The high-molecular-mass bands could be
precipitated by an anti-c-Fos antibody following incubation of
in vitro-translated unlabeled protein in the presence of
[32P]ATP (not shown).
To investigate whether c-Fos-ubiquitin adducts are essential
intermediates in the proteolytic process and to further corrob-
orate the role of the 26S proteasome complex in their degra-
dation, we generated high-molecular-mass conjugates of labeled
c-Fos. The enzymes used for conjugation were inactivated by
the addition of NaOH followed by neutralization with HCl.
The conjugates were then subjected to proteolysis in the pres-
ence of purified 26S proteasome complex (56). c-Fos conju-
gates were degraded in an ATP-dependent manner by purified
26S proteasome complex. The unmodified protein was not
recognized by the 26S proteasome (data not shown). The per-
cent degradation was 0% without 26S or ATP, 4% 6 0.2% with
26S and without ATP, and 48% 6 2.7% with 26S and ATP
(assayed in duplicate; range shown).
Identification of the ubiquitin-protein ligase E3, involved in
c-Fos conjugation. Molecular analysis of the complex mecha-
nisms of degradation of c-Fos requires purification and char-
acterization of the enzymes involved. We have shown (Fig. 2)
that the recently described ubiquitin carrier enzyme E2-F1 (2)
is essential for c-Fos conjugation. Unlike most members of the
E2 family, this protein did not adsorb to the anion-exchange
resin at neutral pH and was purified from the unadsorbed
material (fraction I). The enzyme is not specific to c-Fos and is
involved in the degradation of a variety of substrates, p53, for
example (9, 50, 51). In a previous study, Scheffner and col-
leagues demonstrated that the E2 enzyme transfers ubiquitin
from E1 to a cysteine moiety in the E3 molecule E6-AP (E6-
associated protein), involved in conjugation of p53 (52). The
activated ubiquitin residue is then transferred to the substrate
that is specifically bound to E3 (7, 52). For initial character-
ization of the E3 protein involved in recognition of the c-Fos
transcription factor, we first tested whether the wheat germ
extract used for in vitro translation of the labeled substrate
contains the ligase. Ubiquitin conjugates could not be detected
upon incubation of the substrate alone (not shown) or follow-
ing addition of purified E1 and E2-F1 (Fig. 6A, lane A). In-
creasing the concentration of the wheat germ extract yielded a
similar negative result. We concluded that the E3 enzyme
required for c-Fos conjugation was not present in the wheat
germ extract. It should be noted that E6-AP, the E3 enzyme
involved in p53 conjugation, is also absent from the wheat
germ extract (51). We observed that the E3 protein necessary
for ubiquitination of c-Fos is present in fraction IIA (Fig. 6A,
lane B; data for other reticulocyte lysate subfractions not
shown).
To further characterize the ligase involved, we resolved re-
ticulocyte lysate fraction IIA by gel filtration chromatography
over a Superdex 200 column. Samples from the concentrated
7112 STANCOVSKI ET AL.
FIG. 7. Scheme for purification of E3-Fos. Reticulocyte lysate was applied to
a DEAE-cellulose resin and further resolved as described in Materials and
Methods.
fractions were analyzed for formation of [35S]methionine-la-
beled c-Fos conjugates in the presence of purified E1 and
E2-F1 enzymes (Fig. 6A). In parallel, we examined the same
fractions for conjugation of [35S]methionine-labeled p53 in the
presence of unlabeled, in vitro-translated E6 (Fig. 6B). As
depicted in Fig. 6A, c-Fos-conjugating activity peaked in frac-
tions 25 and 26, corresponding to an apparent molecular mass
of ;260 to 280 kDa. In striking contrast, p53-conjugating ac-
tivity peaked at fraction 28, corresponding to a molecular mass
of ;120 kDa. This activity represents, most probably, E6-AP
(30). In addition, a truncated, bacterially expressed form of
E6-AP that is active in p53 conjugation (a generous gift from
Martin Scheffner) (50) was inactive in c-Fos conjugation (data
not shown). Purified E3a (46) was also inactive in the presence
of both 14-kDa E2 (the N-end rule E2, the mammalian ho-
molog of the yeast UBC2/RAD6 protein [33]) and E2-F1 (not
shown; see also below). The c-Fos-conjugating ligase is also
different from the N-end rule E3b (22) because it does not
interact with 14-kDa E2 (not shown). Taken together, these
findings clearly indicate that the enzyme that conjugates c-Fos
is a novel protein distinct from the several known ligases in-
volved in conjugation of p53 and N-end rule substrates.
Purification of the ubiquitin ligase E3-Fos. The initial iden-
tification of a novel E3 activity involved in c-Fos conjugation to
ubiquitin (designated E3-Fos) prompted further purification
and characterization of the protein. Reticulocyte lysate was
used as a source for the enzyme. The purification scheme (Fig.
7) involved a combination of anion-exchange (DEAE-cellu-
lose, Pharmacia Resource, and Mono Q), gel filtration (Super-
dex 200 HR), hydrophobic (Bio-Rad Methyl-HIC), and hydrox-
ylapatite (Bio-Rad HTP) chromatographies. Column fractions
MOL. CELL. BIOL.
were assayed for E3 activity based on their ability to conjugate
ubiquitin to [35S]methionine-labeled c-Fos in the presence of
purified E1 and E2-F1 enzymes. In parallel, the protein profile
of the different fractions isolated during purification was mon-
itored by SDS-PAGE and Coomassie brilliant blue staining.
The peak of activity eluted from all columns comigrates (in
SDS-PAGE) with a protein of ;140 kDa. Figure 8 presents the
peak of activity recovered from the last two purification steps
along with the profiles of the proteins resolved by electro-
phoresis. It is clear that the indicated 140-kDa protein eluted
from the different resins comigrates with the activity. It was
important to follow the activity and protein profile in all col-
umns, as the E3 enzyme has not been purified to homogeneity.
Taken together, the results strongly suggest that the 140-kDa
protein is indeed E3-Fos (see also below). A summary of the
purification procedure is given in Table 1: 0.1 mg of protein
was isolated, with an overall purification of ;335-fold and a
recovery of ;2.0%. An attempt to sequence the protein from
the N-terminal residue failed, suggesting that the molecule is
blocked at this residue. Eight peptides derived from tryptic
hydrolysis of the E3-Fos enzyme were subjected to Edman
degradation, and their sequences revealed no significant ho-
mology with any known protein (not shown). Cloning of the
gene and expression of the protein will be essential for un-
equivocal identification of the enzyme as the c-Fos ligase and
for a detailed analysis of the mechanism of action of this novel
E3 protein.
Characterization of E3-Fos ubiquitin-protein ligase. Ubiqui-
tination of c-Fos involves the concerted action of the ubiqui-
tin-activating enzyme E1, the ubiquitin-conjugating enzyme
E2-F1, and a novel ubiquitin-protein ligase, E3. Using gel
filtration chromatography, we have demonstrated that the E3
protein has an apparent molecular mass of ;280 kDa. It is
probably a homodimer, as the molecular mass of the SDS-
PAGE-resolved protein is ;140 kDa. The polypeptide chains
of the dimer are not linked via -S-S- bonds, as electrophoresis
of the protein in a denaturing environment but in the absence
of b-mercaptoethanol revealed the same 140-kDa monomer.
We investigated the possibility that E3-Fos, like E6-AP (52),
contains an active sulfhydryl (SH) group involved in ubiquitin
binding and transfer. As can be seen in Fig. 9, the activity of the
enzyme was inhibited significantly by the alkylating agent N-
ethylmaleimide. To demonstrate that the 140-kDa protein is
indeed the E3 enzyme and to further corroborate the role of
the SH group in mediating ubiquitin transfer, we generated a
thioester of the enzyme with labeled ubiquitin in the presence
of E1, E2, and ATP (2, 20). As shown in Fig. 10, incubation of
E3-Fos in the presence of the two other enzymes leads to the
formation of a thiol group-sensitive adduct between ubiquitin
and the ligase. This product had a molecular mass of ;160 kDa
(Fig. 10, lane 5). Addition of excess free thiol groups (b-
mercaptoethanol) results in the disappearance of most of the
radioactivity (Fig. 10, lane 6). The remaining label migrated at
;150 kDa. This SH-insensitive compound is probably an
autoubiquitination product of the enzyme. This hypothesis can
only be tested following cloning and expression of the enzyme.
Not surprisingly, the E1-ubiquitin thioester was also labile to
the addition of the reducing agent (Fig. 10, lanes 1 and 3). We
were able to detect the E2-ubiquitin adduct (lane 2) only fol-
lowing a shorter exposure. It is ‘‘hidden’’ in the gel shown by
the labeled ubiquitin.
It was important to further examine the specificity of recog-
nition of the novel E3 enzyme towards different substrates. The
initial identification of the E3-Fos protein indicated that the
activity is distinct from E6-AP, the ligase required for conju-
gation of p53, and from the N-end rule E3s, E3a and E3b (see
VOL. 15, 1995 c-Fos DEGRADATION BY THE UBIQUITIN SYSTEM 7113

FIG. 8. Comigration of c-Fos-conjugating activity with the putative 140-kDa E3-Fos. E3-Fos activity in the various fractions was determined by monitoring ubiquitin
conjugation to [35S]methionine-labeled c-Fos in the presence of E1 and E2-F1 enzymes as described in Materials and Methods. Conjugation was quantitated by
phosphorimaging plate scanner analysis and expressed as a percentage of total radioactivity incorporated into high-molecular-weight adducts. In parallel, samples from
the same fractions were resolved by SDS–7.5% PAGE, and the proteins were visualized by staining the gel with Coomassie brilliant blue. (A) Conjugation activity of
E3-Fos resolved on a hydrophobic (Methyl-HIC) resin. Peak fractions from the Superdex 200 gel filtration chromatography column were processed and resolved on
a Methyl-HIC column as described in Materials and Methods. Aliquots (2.5 ml) from the concentrated fractions were monitored for c-Fos conjugation. (B)
Electrophoretic analysis of E3-Fos-containing fractions resolved on the Methyl-HIC column. Aliquots (10 ml) were electrophoresed on a 7.5% polyacrylamide gel,
followed by staining with Coomassie brilliant blue. Lane M, molecular size markers. Lanes 11 to 19 represent the corresponding gradient fractions as described in
Materials and Methods. (C) Conjugation activity of E3-Fos resolved on hydroxylapatite (HTP). The activity was determined as described in the legend to panel A. (D)
Electrophoretic analysis of E3-Fos-containing fractions resolved on the HTP column. Aliquots from the corresponding fractions were processed as described in the
legend to panel B. The gel was stained with silver.

above). Accordingly, E3-Fos could not promote the conjuga-
tion of lysozyme and oxidized RNase A (E3a substrates) or of
protein S (E3b substrate) (not shown). Also, E3a could not
substitute for E3-Fos in c-Fos conjugation (see above). Con-
jugation of other substrates, such as histone H2A, actin, tro-
ponin T, and MyoD, could not be stimulated by this ligase
either. These substrates are conjugated by a distinct and novel
E3 enzyme (17a). Interestingly, E3-Fos is involved in ubiquiti-
nation of c-Jun, the other component of the AP-1 transcription
factor complex (not shown). This finding raises the possibility
that both components of the complex are targeted by the same
machinery.
Conjugation recognition signal(s) in c-Fos. It was previously
demonstrated that whereas the t1/2 of c-Fos in vivo is ;30 min,
the v-fos gene product is more stable and has a half-life of ;2
h (12). Interestingly, c-Jun does not affect v-Fos stability (45).
Nucleotide sequence analysis revealed that the v-fos gene dif-
fers from c-fos in that it has undergone an out-of-frame dele-
tion of 104 bases (60, 61). Consequently, the v-Fos protein has
49 amino acids at its C terminus that are unrelated to the 48
amino acids at the C terminus of the c-Fos protein. In partic-
ular, the modification affected serine residues necessary for
phosphorylation. As a result, the extent of phosphorylation of
v-Fos is markedly decreased. We tested the ability of v-Fos to
be conjugated to ubiquitin. As can be seen in Fig. 11, conju-
gation of v-Fos is much less efficient than that of c-Fos (about
threefold) both in a crude lymphocyte extract (Fig. 11A) and in
a reconstituted system containing the purified conjugating en-
zymes (Fig. 11B). Not surprisingly, v-Fos is also stable in the
cell-free system (45), and ;80% of the protein can be detected
following 3 h of incubation (not shown).
DISCUSSION
Many regulatory cellular proteins are extremely unstable. In
the case of signal-inducible transcription factors, it is apparent
that changes in their steady-state levels can influence the ac-
tivity of the corresponding target genes. Thus, it is important to
understand the molecular mechanisms that govern the cellular
stability of these proteins.
7114 STANCOVSKI ET AL. MOL. CELL. BIOL.

TABLE 1. Summary of the purification of E3-Fos ligasea

Total Total Sp act Purification Recovery
Fraction
protein (mg) activity (U) (U/mg) (fold) (%)

Fraction IIA 1,540 56,980 37 1 100

Resource Q 76 34,200 450 12.2 60
Superdex 200 25 16,175 647 17.5 28.3
Methyl-HIC 3.25 3,120 960 25.9 5.4
HTP 0.1 1,240 12,400 335.1 2.1
a
Two liters of reticulocyte lysate was used for purification. The protein con-
centration was determined by the Bradford method (3). Activity was determined
by measuring conjugation of [35S]methionine-labeled c-Fos in the presence of
E1, E2-F1, and the tested fractions as described in Materials and Methods.
Quantitation was performed with a phosphorimaging plate scanner and is ex-
pressed in units of activity. One unit represents incorporation of 1% of radio-
activity into conjugates per milligram of protein (determined in the linear range
of the reaction). The resins used for the purification are: Resource Q, anion-
exchange resin (Pharmacia); Superdex 200 HR, gel filtration matrix (Pharmacia);
Methyl-HIC, hydrophobic matrix (Bio-Rad); and HTP, hydroxylapatite beads
(Bio-Rad).
In this study, we demonstrated that the degradation of the
c-Fos transcription factor is mediated by the ubiquitin system
both in vivo and in vitro. Using a cell cycle-arrested mutant
that harbors a thermolabile ubiquitin-activating enzyme, E1,
we show that c-Fos is stabilized at the nonpermissive temper-
ature. In contrast, the protein is unstable at the permissive
temperature and at both temperatures in the wild-type cell. A
mutant cell line that was rescued by transfection with a cDNA
encoding the wild-type E1 enzyme displays a wild-type pheno-
type. Using the same strategy, Chowdary and colleagues (6)
demonstrated the involvement of the ubiquitin system in the
degradation of p53. However, it should be noted that in intact
cells, it is difficult to determine whether the E1 enzyme is
directly involved in c-Fos conjugation and degradation or
whether inactivation of the enzyme affects a different protein
that resides upstream in the proteolytic pathway of c-Fos. For
example, E1 may be involved in the degradation of an uniden-
tified specific proteolytic inhibitor or an ancillary protein that
stabilizes c-Fos: stabilization of the inhibitor or protein leads to
stabilization of the target protein. Another alternative may
involve targeting of c-Jun. The Jun protein, which is the other
component of the AP-1 transcription complex, is also targeted
for degradation following ligation of ubiquitin (59). It is pos-
sible that c-Jun is degraded first and thus exposes, in a second-
ary manner, the c-Fos protein to the action of the ubiquitin
system or a ubiquitin-independent pathway. However, it was
recently reported that c-Jun can be efficiently degraded by the
26S proteasome without prior ubiquitination (31). To further
corroborate the role of ubiquitination in targeting c-Fos and to
FIG. 10. Formation of a thioester adduct between ubiquitin and purified
E3-Fos. The reactions were performed as described previously (2) and in Ma-
terials and Methods. The samples contained E1 and 125I-ubiquitin alone (lanes
1 and 3) or with E2-F1 (lanes 2 and 4) or with E2-F1 and purified E3-Fos (lanes
5 and 6). Reaction mixtures were incubated at 378C for 20 min and divided into
two equal portions that were resolved via SDS-PAGE in the presence (1) or
absence (2) of b-mercaptoethanol (b-ME; 0.1 M), as indicated. The E1- and
E3-ubiquitin thioester adducts are indicated (E1-S;Ub and E3-S~Ub, respec-
tively), as is the unreacted radioactive ubiquitin (125I-Ub). E3F-Ub?, putative
monoubiquitinated product of the E3-Fos ligase.
complement the in vivo findings, it was necessary to establish a
cell-free proteolytic system and study the different enzymes
and ancillary components involved.
We were able to reconstitute the c-Fos degradation pathway
in vitro and to demonstrate that conjugation of ubiquitin to
c-Fos is an essential intermediate step in the proteolytic pro-
cess. c-Fos is tagged by ubiquitin (Fig. 2), and the formation of
high-molecular-mass adducts is inhibited by MeUb (Fig. 3),
suggesting that targeting of the protein requires multiple ubi-
quitination. It is clear that these adducts serve as essential
intermediates in the process, since only the conjugated protein
is recognized by the 26S proteasome and degraded (see above
and Fig. 5). Addition of c-Jun increases both conjugation and
subsequent degradation of c-Fos (Fig. 4). This is probably due
to a physical interaction between the two proteins (45). It
should be noted that both conjugation and degradation pro-
ceed in the absence of c-Jun. However, the two processes,
proteolysis in particular, are stimulated significantly following
addition of the Jun protein. It is possible that the effect of
c-Jun on conjugation is greater than the one that we detect and
that the conjugates formed are rapidly deubiquitinated by

FIG. 9. E3-Fos ubiquitin-ligase has an active SH group. Conjugation of ubiq-

uitin to c-Fos was monitored as described in Materials and Methods. The reac-
tion mixtures contained all three conjugating enzymes together with ATP-g-S.
For alkylation of SH groups, the indicated enzyme was incubated for 10 min at
228C in the presence of N-ethylmaleimide (NEM; 10 mM). The reagent was
neutralized by the addition of 6 mM DTT. Lane 1, complete reaction mixture; FIG. 11. Conjugation of ubiquitin to c-Fos and v-Fos proteins. Conjugation
lane 2, the E2-F1 enzyme pretreated with NEM and neutralized with DTT; lane reactions of the labeled substrates were performed as described in Materials and
3, E3-Fos pretreated with NEM and neutralized with DTT; lane 4, E3-Fos Methods. (A) Conjugation in crude lymphocyte extract. (B) Conjugation in a
pretreated with NEM that was previously incubated in the presence of DTT. Ori, reconstituted system containing E1, E2-F1, and E3-Fos (derived from the Mono
origin of the gel; conj, conjugates. Q purification step [Fig. 7]; 0.75 mg). Lanes 1, c-Fos; lanes 2, v-Fos.
VOL. 15, 1995
isopeptidases that are insensitive to ubiquitin aldehyde. The
involvement of Jun represents yet another example of trans
recognition and targeting. The tumor suppressor protein p53 is
targeted for degradation by the human papillomavirus onco-
protein E6 (53). Ornithine decarboxylase is targeted for deg-
radation by the 26S proteasome complex without prior ubiqui-
tination in a process that requires antizyme (39). However, the
case of c-Fos–c-Jun dimer differs from the other known cases
of trans-targeted degradation. In the p53-E6 complex, as well
as in the ornithine decarboxylase-antizyme complex, one sub-
unit of the binary complex provides the recognition site and
remains stable, whereas the other is targeted for degradation.
In the c-Fos–c-Jun heterodimer, both components appear to
be ubiquitinated. However, no evidence is available concerning
the degradation of c-Jun adducts. Also, the Jun protein is an
integral part of the AP-1 transcriptional complex and essential
for its activity. This is not the case with E6 and antizyme.
Therefore, it is expected that c-Jun is involved in regulating the
stability of the complex.
Dissection of the enzymatic components involved in c-Fos
conjugation and degradation reveals that conjugate synthesis is
catalyzed by the ubiquitin carrier protein E2-F1 and its human
homolog UbcH5 (Fig. 2). The 14-kDa E2, the N-end rule
ubiquitin-activating enzyme, is significantly less active. This is
evident from the low conjugating activity of fraction II, which
contains the enzyme (Fig. 2B and C). Ubiquitination requires
an additional novel protein that presumably functions as a
ubiquitin-protein ligase, E3 (Fig. 6). Purification of the novel
E3 activity revealed that the enzyme is a homodimer with an
apparent molecular mass of ;280 kDa (Fig. 6 and 8). The
protein contains an active SH group that, like the E6-AP en-
zyme, functions in the thioester cascade involving transfer of
ubiquitin from E1 to E2-F1, from E2-F1 to E3-Fos, and then to
the substrate (Fig. 9 and 10). E3-Fos is essential for conjuga-
tion, suggesting involvement in recognition of the substrate.
The recognition appears to be specific, as the novel E3 does
not conjugate the tumor suppressor protein p53 and a variety
of N-end rule substrates and other proteins, such as actin,
histone H2A, troponin T, and MyoD. We suspect, however,
that the enzyme is not entirely specific to c-Fos and that other
substrates will be discovered. We have already shown that it
can conjugate c-Jun (not shown). An attractive model is that a
single E2 enzyme interacts with a subset of E3s that recognize
common structural motifs on their protein targets. E2-F1 is
involved in the conjugation of both p53 (9, 50, 51) and c-Fos
(this study), but the E3 enzymes involved are clearly distinct.
The different E3 enzymes may not be specific to a single sub-
strate, and it is likely that both E3-Fos and E6-AP recognize
other proteins as well. As mentioned, our data suggest the
involvement of E3-Fos in c-Jun conjugation.
The mode of recognition of c-Fos by this E3 enzyme is not
yet clear. In certain cases, specific phosphorylation or dephos-
phorylation appears to target proteins for degradation. IkBa is
targeted for degradation by the ubiquitin system following
signal transduction-induced phosphorylation of Ser-32 and
Ser-36 (4, 57). Mos is targeted for degradation by specific
dephosphorylation of Ser-3 (41). In the case of Fos, it has been
demonstrated that the product of the viral oncogene v-fos is
more stable than the cellular normal protein and that c-Jun
does not affect its stability (45). The two proteins vary in the
C-terminal domain, which contains multiple phosphorylation
sites. We have shown that v-Fos is recognized less efficiently by
the ubiquitin-conjugating machinery.
We could not rule out completely the partial involvement of
another proteolytic system(s) in the breakdown of c-Fos. For
example, it has been suggested that both c-Jun and c-Fos are
c-Fos DEGRADATION BY THE UBIQUITIN SYSTEM 7115
substrates for a Ca21-dependent calpain (5, 27). Our results, as
well as those of Treier and colleagues (59) studying the deg-
radation of c-Jun, indicate that the ubiquitin system is the main
pathway that controls c-Fos and c-Jun stability.
Control of stability of oncogene products and tumor sup-
pressors frequently contributes to transformation. Our results
provide additional evidence for the involvement of the ubiqui-
tin system in the breakdown of a key cellular regulator.
ACKNOWLEDGMENTS
We thank Dirk Bohmann (European Molecular Biology Laboratory,
Heidelberg, Germany) for providing the pKH6 plasmid; Julian Adams
and Ross Stein (MyoGenics, Inc., Cambridge, Mass.) for the protea-
some inhibitor MG115; Martin Scheffner (Deutsches Krebsforschungs-
zentrum, Heidelberg, Germany) for the kind gift of the UbcH5 clone;
and Patricia M. Handley-Gearhart (Washington University School of
Medicine, St. Louis, Mo.) for the ts-E1 cells. We also thank Dganit
Shkedy and Beatrice Bercovitch for help and advice. We are grateful
to Daniel Schindler (The Weizmann Institute of Science, Rehovot,
Israel) for critically reading the manuscript.
This research was supported by grants from the Council for Tobacco
Research, Inc. (CTR), German-Israeli Foundation for Scientific Re-
search and Development (G.I.F.), Israeli Academy of Humanities and
Sciences, Israel Cancer Research Fund (ICRF, USA), Israel Cancer
Society, Foundation for Promotion of Research at the Technion, and
the Henri Gutwirth Fund for the Promotion of Research. I.S. was
supported by a fellowship from the Lady Davis Foundation.
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